Significant variations in the metabolism of various drugs and environmental chemicals which are metabolized via cytochrome P450 (CYP) enzymes exist between humans. Many of these interindividual variations are attributed to polymorphisms in the CYP2C subfamily of enzymes. Current work focused on investigating the functional significance of specifically bioengineered CYP enzymes towards the metabolism of various substrates. Human recombinant CYP2C proteins for 2C8, 2C9, 2C18, and 2C19, were expressed yeast. Various CYP2C mutants, and CYP2C9/CYP2C19 chimeras containing various substrate binding sites were also constructed. Results from these studies indicated that hepatic CYP2C19 protein is the major high affinity 4-hydroxylase of S-mephenytoin. Metabolism of the antiulcer drug omeprazole by these human recombinant enzymes in vitro revealed that several enzymes including CYP2C19, CYP3A, and CYP2C8 were able to hydroxylate omeprazole. However, kinetic analysis revealed that CYP2C19 was the only high affinity enzyme and it would be expected to contribute the most to omeprazole hydroxylation at therapeutic doses. Further, CYP3A4 was found to be the principal enzyme responsible for omeprazole sulfone formation. Present work further demonstrated that the Leu359 variant of 2C9 metabolizes the antidiabetic drug tolbutamide with a lower affinity than the predominant Ile359. We also assessed the metabolic activities of various chimeras of CYP2C9/CYP2C19 which were constructed using site-directed mutagenesis to determine the functional importance of individual amino acids which result in variations in substrate specificity for the CYP2C enzymes. Residues 99, 220, and 221 of human CYP2C19 were identified as key determinants of omeprazole hydroxylase activity.